This invention relates to a process for producing interferon inducers (hereinafter referred to as IF inducers) originating from the tissues of plants. Interferon, hereinafter also referred to as IF, is a substance capable of acting upon animal or human cells to inhibit the growth of a virus and is a type of protein liberated from the cell in response to viral infection. The activity of IF is specific with respect to an animal species and non-specific with respect to a viral species and may vary, with diferring conditions used for its induction. It is also known that the growth of certain animal tumour type viruses may significantly be inhibited by IF under certain conditions. A substance capable of acting upon animal or human cells to induce IF is designated as an IF inducer. Thus an IF inducer is of potential interest in the prevention and treatment of various human and animal diseases caused by viral infection. However, various known IF inducers have never been used in practice for such a purpose because of certain defects.
With regard to IF inducers originating from the plant tissues, it was known that certain mitogenic agents such as phytohemagglutinin (PHA) [Wheelock, Science, 140:310 (1965) and J. Biol. Chem., 212:607-615 (1955)], pokeweed mitogen [Friedman et al, Proc. Soc. Exp. Med., 125:90 (1967) and J. Exp. Med., 124:859-872 (1966)] and concanavalin A [Willen et al, Cell. Imminol., 6:110 (1973) and Methods of Carbohydrate Chemistry, vol. VI, 108-110 (1972)] respectively isolated from the tissues of kidney bean, poke weed and horse bean have an extremely low IF inducing activity. These mitogenic agents have been prepared by extracting the active substance with a saline solution or buffer solution from the plant tissues, treating the extracted solution with an alcohol to give a precipitate, subjecting the precipitate to column chromatography. Due to their extremely low IF-inducing activity, no successful attempt has been made to use these mitogenic agents for preventing and treating various diseases caused by viral infection. Other IF inducers orginating from the plants are also known. That is, Kojima (one of the coinventors of the present invention) et al [Japanese Patent Application as laid open to public inspection as Kokai Koho No. 32107/78 ] disclosed an IF inducer which is believed to be a kind of heteropolymeric saccharides containing as main constituents hexose (40%), protein (5%) and uronic acid (5%) having a molecular weight of more than 100,000. This substance is isolated from the root of Angelica acutiloba Kitagawa (known in Japan as Toki) by extracting the root with hot water to give an extracted solution, subjecting the same to dialysis to give a residue, adding acetone to the residue to give precipitate, followed by freeze-drying the same. If desired, the extracted solution may be made up to a suitable quantity before dialysis by concentrating in vacuo or by using a Diaflo membrane (MW=10.000). Subsequently, Kojima and Tamamura [Japanese Patent Application as laid open to public inspectionas Kokai Koho No. 99313/78] disclosed an IF inducer having a molecular weight of more than 20,000 (mainly more than 60,000) and containing as main constituents a 1-3 bonded glucose (hexose: more than 90%). This IF inducer is prepared by extracting the peeling of a mulberry e.g. Morus alba Linne or M. bombycis Koidzumi with hot water, adding an organic solvent to the extracted solution to give precipitate, adding to the same a small amount of water, subjecting the mixture to dislysis to give a residue and freez-drying the residue. If desired, the extracted solution may be made up to a suitable quantity by concentrating in vacuo or by using a Diaflo membrane. These two IF inducers are soluble in water and insoluble in organic solvents and have high IF-inducing activity.
As a result of further studies, it has been found that certain substances which we have isolated from the tissues of various higher plants show high IF-inducing activity. The present invention is based on the discovery that various plants which have been used as traditional remedies in Japan and China are not only effective in regulating general health but are also believed to be effective in curing common cold. However, there has never been any disclosure that these traditional Sino-Japanese remedies contain a substance having IF-inducing activity except the above-mentioned Angelica acutiloba and mulberries discovered by Japanese researchers.
We have investigated very many plants of various species and have found that certain plants are capable of producing IF-inducers. Thus, the essence of this invention resides in the selection or discovery of the plants capable of producing IF-inducers.
According to this invention, there is provided a process for producing an IF inducer, characterized in that a water-soluble IF inducer is extracted with water from the tissue of a plant selected from the genera Atractylodes, Lonicera, Plantago, Lithospermum, Ligusticum, Cnidium, Bupleurum, Notopterygium, Heracleum, Aralia, Panax, Polygala, Sophora, Euchresta, Astragalus, Sinomenium, Stephania, Cocculus, Cimicifuga, Rheum, Gastroida, Asparagus, Pinellia, Evoida and variants thereof capable of producing said IF inducers and said IF inducer is recovered from the extract thereby obtained.
The plants which may be used for the purpose of this invention are shown in the following Table 1. They are harvested usually in autumn, although, for example, the roots may be used at any time. The term "variant" used herein denotes a naturally or artificially occuring hybrid or mutant of the parent plant. Preferred plants are indicated in the following Table 2. In Table 2, the weight indicated below the item "Tissue" in the bramcket denotes a dry weight of the crude product obtained by ultrafiltration using a membrane capable of fractionating substances having a molecular weight of more than 50,000 and is calculated on the basis of 100 g (dry weight) of the plant tissue used.
In this specification, the botanical names are indicated by referring to the following publications:
(1) John D. Keys: Chinese Herbs, Their Botany, Chemistry and Pharmacodynamics (1976). pages 40-41, 56-57, 84-87, 114-115, 124-125, 136-137, 148-149, 152-153, 158-159, 178-179, 194-195, 212-213, 268-269, 290-291. PA0 (2) Richard Hyatt: Chinese Herbal Medicine, Ancient Art and Modern Science (1978), pages 112-115, 120-127, 130-131, 134-135, 136-137, 140-141. PA0 (3) Ohwi: Flora of Japan (1965): published by Shibundo, Tokyo in Japanese version, pages 419-420, 960-961, 1002-1003, 1368. PA0 (4) North American Flira, vol. 24 (1926), published by The New York Botanical Garden, U.S. PA0 (5) Illustrated Flora of the Pacific States, vol. 1, (1940), vol 2 (1950), vol. 3 (1951), vol. 4 (1965), published by The Stanford University Press, U.S. PA0 (6) Colored Illustration of Herbaceous Plants of Japan (1980) published by Hoiku-sha, Osaka, Japan in Japanese version. PA0 (7) Flora Europaea, vol. 2 to vol. 4 (1968), vol 5 (1976) published by Cambridge University Press, U.K.
TABLE 1 ______________________________________ Family Molecular** (Subfamily) Genus Tissue* weight(.times. 10.sup.4) ______________________________________ Compositae Atractylodes Rhizome 5-100 Caprifoliaceae Lonicera Flower 5-100 Plantaginaceae Plantago Seed & Leaf 5-50 Borraginaceae Lithospermum Root 5-50 Umbelliferaceae Ligusticum Root 5-50 Cnidium Rhizome 5-50 Bupleurum Root 5-50 Notopterygium Root 5-100 Heracelum Root 5-100 Angelica Root 5-100 Araliaceae Aralia Root 5-50 Panax Root 5-50 Polygalaceae Polygala Root 5-50 Leguminosae Sophora Root 5-50 Euchresta Root 5-50 Astragalus Rhizome 5-100 Menispermaceae Sinomenium Root 5-100 Stephania Root 5-100 Cocclus Root 5-100 Ranunclaceae Cimicifuga Rhizome 5-50 Polygonaceae Rheum Rhizome 5-100 Orchidaceae Gastrodia Rhizome 5-50 Liliaceae Asparagus Rhizome 5-100 Araceae Pinellia Rhizome 5-100 Rutaceae Evoida Fruit 5-50 ______________________________________ Notes: *Tissue where IF inducer is rich. **Approximate range where IF activity is prevelent.
TABLE 2 __________________________________________________________________________ Preferred plants and IF induction in vitro and in vivo In vitro Example (Family) (.mu.g/ml) No. Botanical name Tissue 10 1.0 0.1 In vivo __________________________________________________________________________ (Compositae) Atractylodes 6 ovata DC rhizome &gt;100 88 30 105 .+-. 35 (1.06 g) 7 japonica Koidz. rhizome &gt;100 92 45 145 .+-. 45 (1.13 g) lancea DC rhizome &gt;100 80 40 120 .+-. 40 (0.98 g) chinensis DC lancea DC var. simplicifolia Kitam. (Caprifoliaceae) Lonicera 8 japonica Thunb. flower &gt;100 &gt;100 40 95 .+-. 25 (1.5 g) 9 confusa DC flower &gt;100 90 35 80 .+-. 20 (1.4 g) chinensis Watson 10 maaeckii Maxim flower &gt;100 95 38 85 .+-. 30 similis Hems tragophylla Hemsl pampaninii Levl hypoglauca Miq Macranthoides Hand-Mazz affinis Hook at Arn. flexuosa Thunb brachypoda DC chrysantha Turcz demissa Rehd morrowii A. Gray linderifolia Max strophiophora Fr. praeflorens Batalin ramosissima Fr caprifolium L. xylosteum L. sempervirens Ait (Plantaginaceae) Plantago 11 asiatica L. seed 52 &lt;10 &lt;10 32 .+-. 12 (0.8 g) 12 leaf &gt;100 &gt;100 50 190 .+-. 65 (1.3 g) 13 patagonica leaf &gt;100 &gt;100 45 185 .+-. 35 (1.2 g) 14 japonica Fr leaf &gt;100 &gt;100 45 175 .+-. 40 (1.3 g) lanceolata L. mohnikei Miq camtschatica Cham virginica L. major L. psyllium L. loureiri Roem et Schult (Borraginaceae) Lithospermum erythrorhizon Sieb root &gt;100 &gt;100 70 230 .+-. 30 et Zucc (1.1 g) euchromum Royle root &gt;100 &gt;100 80 220 .+-. 35 (1.2 g) arvense L. 17 officinale L. root &gt;100 &gt;100 75 225 .+-. 40 (1.3 g) officinale var. erythrorhizon Max zollingeri DC ruderale Dougl. (Umbelliferaceae) Ligusticum 18 officinale Kitagawa rhizome &gt;100 &gt;100 80 180 .+-. 30 (3.2 g) 19 chauxiong Hort rhizome &gt;100 &gt;100 50 150 .+-. 25 (2.8 g) 20 wallichii Fr rhizome &gt;100 &gt;100 55 150 .+-. 30 (3.1 g) japonica Max Cnidium officinale Makino 21 japonica Miq rhizome &gt;100 &gt;100 55 160 .+-. 30 (3.0 g) ajanense Drude tachiroei Makino Bupleurum 22 falcatum L. root &gt;100 &gt;100 85 190 .+-. 32 (1.1 g) 23 longiradiatum Turcz root &gt;100 &gt;100 64 160 .+-. 40 (1.2 g) 24 scorzonaefolium root &gt;100 &gt;100 55 150 .+-. 40 Willd (1.2 g) nipponicum Koso-Poliansky longiradiatum var. shikotanensis Ohwi shikotanensis Hort triadiatum Adams longiradiatum var. brevirsdiatum Fr. Scm Notopterygium 25 incisium Ting root &gt;100 &gt;100 92 200 .+-. 20 (2.2 g) Heracleum lanatum Michx root &gt;100 &gt;100 80 280 .+-. 40 (2.8 g) hemsleyanum Michx canadium Fr. stenopterum Diels (Araliaceae) Aralia 26 cordata Thunb root &gt; 100 90 35 130 .+-. 28 (1.2 g) Panax 27 ginseng Meyer root &gt;100 80 10 25 .+-. 5 (1.2 g) 28 japonica Meyer root &gt;100 55 10 22 .+-. 7 (1.4 g) quinquefolius L. pseudo-ginseng Wall (Polygalaceae) Polygala 29 tenuifolia Willd root &gt;100 &gt;100 78 90 .+-. 20 (1.5 g) 30 japonica Houtt root &gt;100 &gt;100 70 90 .+-. 20 (1.1 g) tatarinowii Regel reinii Fr. et Sav (Legminosae) Sophora 31 angustifolia Sieb et root &gt;100 &gt;100 85 120 .+-. 40 Zucc (2.6 g) angustifolia var. purpurascens Makino flavescens var. angustifolia Kitagawa 32 flavescens Ait. root &gt;100 &gt;100 80 110 .+-. 55 (2.5 g) 33 subprosarata Chun et root &gt;100 &gt;100 90 105 .+-. 45 Chun Euchresta 34 japonica Benth root &gt;100 &gt;100 80 105 .+-. 45 (1.3 g) Astragalus 35 membranaceus Bunge rhizome &gt;100 &gt;100 90 130 .+-. 32 (1.4 g) 36 mongholicus Bunge rhizome &gt;100 &gt;100 80 120 .+-. 40 (1.6 g) adsurgens Pall sinicus L. reflexistipulus Miq. yamamotoi Miyake et al hoantchy Fr (Menispermaceae) Sinomenium 38 acutum Rhed et Wils root &gt;100 &gt;100 65 270 .+-. 30 (1.1 g) diversifoloium Diels Stephania 39 tetrandra Moore root &gt;100 &gt;100 70 250 .+-. 40 (1.3 g) Cocculus 40 trilobus DC root &gt;100 &gt;100 75 210 .+-. 45 (1.1 g) sarmentosus DC laurifolius DC thunbergii DC (Ranunclaceae) Cimicifuga 41 simplex rhizome &gt;100 &gt;100 100 190 .+-. 40 Wormskarl (1.2 g) 42 dahurica Max rhizome &gt;100 80 32 95 .+-. 20 (1.1 g) 43 heracleifolia Komar rhizome &gt;100 75 25 90 .+-. 25 (1.2 g) frigida Royle ternata Miq 44 foetida L. rhizome &gt;100 90 35 100 .+-. 20 (1.0 g) japonica Thunb racemosa Bart (Polygonaceae) Rheum 45 palmatum L. rhizome &gt;100 &gt;100 90 180 .+-. 30 (1.5 g) 46 officinale rhizome &gt;100 &gt;100 50 170 .+-. 30 Baillon (1.4 g) undulatum L. laciniatum L. 47 rhaponticum L. rhizome &gt;100 &gt;100 60 160 .+-. 40 (1.3 g) pontaninii Los- Losinsk. enodi Wall franzenbachii Muent collinianum Baillon speciform Royle (Orchidaceae) Gastroida 48 elata Blume rhizome &gt;100 &gt;100 95 280 .+-. 55 (4.3 g) gracilis Blume nipponica Tuyama acerina Tanaka confusa Hinda et Tuyama (Liliaceae) Asparagus 49 lucidus Lindley rhizome &gt;100 &gt;100 45 130 .+-. 35 (3.5 g) 50 officinalis L. rhizome &gt;100 95 40 120 .+-. 20 (3.3 g) medeoloides Thunb racemosus Willd schberioides Kunth insularis Hance falcatus Benth oligoclonos Max 51 cochinchinensis Merri rhizome &gt;100 &gt;100 35 110 .+-. 30 (3.8 g) (Araceae) Pinellia 52 ternata Breit rhizome &gt;100 &gt;100 60 140 .+-. 35 (4.7 g) tripartita Schott ternata forma angustata Makino 53 tuberifera Tenore rhizome &gt;100 &gt;100 65 150 .+-. 40 (4.9 g) (Rutaeaceae) Evoida 54 rutaecarpa Benth fruit &gt;100 &gt;100 90 330 .+-. 30 (1.4 g) officinale Dude daniellii Hance __________________________________________________________________________
The essence of this invention resides in the discovery or selection of plants capable of producing IF-inducers, As above-mentioned, it was previously known that mitogenic agents from kidney bean, pokeweed and horse bean show a poor IF-inducing activity and that Angelica acutiloba and mulberries are capable of producing IF-inducers. The known IF-inducers from plants are, in general, produced by the following steps: Extraction of plant with water, saline solution or buffer solution.fwdarw.forming a supernatant.fwdarw.fractionation of the supernatant to give a precipitate containing the major portion of the IF-inducing substance by the precipitation method using an alcohol.fwdarw.recovery of IF inducer by freez-drying and/or column chromatography.
As the present IF inducers are soluble in water, insoluble in organic solvents, acidic and high molecular weight substances, it is possible to obtain the desired IF-inducers by any and all known methods. For example, the extraction may be effected with a saline or buffer solution, although the extraction with water is most advantageous with respect to the cost, safety and simplicity of the operation and high purity of the resultant product. Extraction of various useful substances from plants with water are well known and have been made over many years in various countries of the world at a ratio of water to plant, for example, of 30:1 to 5:1 with respect to the extraction economy. However, it is also possible to use a convenient ratio of water to plant, if desired.
It is preferred to use the dried plant for better extraction efficiency and preservation, although it is possible to use the fresh material if desired.
The extraction may conveniently be effected at any temperature from ambient to the boiling point of the extraction mixture for a period sufficient to extract the major portion of the active material in the plant because the present IF inducer is soluble in water. Especially when the extraction is effected upon the seed, a higher temperature e.g. from 80.degree. to 120.degree. C. may be preferred, and in the case of tissues other than the seed a lower temperature e.g. 40.degree. to 75.degree. C. may be preferred. As the extraction may be effected more effectively under alkaline pH, it is preferred to adjust the pH of the water to a pH of 7 to 10 before use by using a conventional alkali such as e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide or a suitable buffer solution. When the extraction is effected at ambient temperature, the extraction may usually be effected for 1 to 5 days, which may be shortened if the extraction temperature is raised. Thus, for example, extraction may be effected for 20 minutes to 6 hours at 45.degree. to 100.degree. C. In this manner, it is possible to extract the major portion of the active substance contained in the plant tissue (in some cases, more than 90%). However, for example, in the cases of extraction of the plants of the genera Bupleurum, Notopterygium, Heracleum, Aralia, Polygala, Evoida, Sterllaria, Gastrodia and the like, it is preferred to extract them at a relatively higher temperature e.g. at 80.degree.-120.degree. C. for 1-2 hours. When the extraction is effected at room temperature with water for a longer period of time, a suitable antiseptic agent may, if desired, be added to the extracting water before use. The extraction may be effected intermittently or continuously at any convenient ratio of water to raw material used, for example, 5:1 to 20:1. As the extraction of water-soluble substance from the plant tissue with water is well known in the art, it is possible to extract the major portion of the active substance from the plant tissue without difficulty. The residue of the raw material is removed from the extracted solution in conventional manner, for example, by presseing, filtering, centrifuging and the like. After this, undesired impurities are removed from the resultant supernatant in order to allow recovery of the active substance.
Although the physico-chemical properties of the IF inducers extracted from various plants have not yet been completely clarified, they are water-soluble and are believed to be high molecular weight substances. According to one embodiment of this invention, the supernatant may be fractionated by ultrafiltration e.g. using a membrane capable of fractionating substances having a molecular weight of at least 50,000, which may be effected e.g. at a pressure of from 0.1 to 5 kg/cm.sup.2. The resultant active fractions are collected, combined and freeze-dried to obtain a crude powder.
It is alternatively possible to recover the active substance from the supernatant by addition of a suitable hydrophilic organic solvent which is miscible with water and incpable of dissolving the active substance such as, e.g. methanol, ethanol, propanol, butanol, acetone and the like at an appropriate concentration (e.g. 40-90% w/v) so as to form a precipitate containing the major portion of the active substance present in the supernatant, which is then dried to give a crude powder. Instead of the organic solvents, it is also possible to use an ammonium salt such as e.g. ammonium chloride, ammonium sulfate, cethylmethylammonium bromide and the like or an inorganic metallic salt such as e.g. zinc chloride, copper chloride and the like at an appropriate concentration (e.g. 20-50% w/v) to form a precipitate which is then desalted in conventional manner, for example, by dialysis, followed by freeze-drying to obtain a crude powder.
The recovery by ultrafiltration is, in general, preferred because on one hand, it is simpler, safer and cheaper in operation and on the other hand, it avoids undesired chemical changes in the desired product. It has been found that by ultrafiltration, more than 90% of impurities such as low molecular weight plant pigments which are sometimes toxic may, in general, easily be removed. Moreover, the fractionation and recovery may be performed in one throuput step.
The resultant crude powder may, if desired, further be purified by a sutable method conventionally used for purifying water-soluble, acidic, high molecular weight substances, for example, by column chromatography using a suitable agent for gel filtration or an ion exchanger. In the former case, the elution may be effected with water, although it is possible to use a suitable buffer solution. In the latter case, the elution may be effected by using a suitable buffer solution.
Prefeered agents for gel filtration are exemplified by Sephadex G-50 to G-200, Sepharose 2B to 6B, Sephacryl S-200 or S-300 (commercial products of Pharmacia Fine Chemicals AB., Sweden), Bio-Gel P-30 to P-300, Bio-Gel A (commercial products of Bio-Rad Laboratories Ltd., U.S.), Sagavac (commercial product of Saravac Laboratories Ltd., U.K.) and the like, and preferred agents for ion exchange treatment are exemplified by DEAE Sephadex A-25 and A-50 (Cl.sup.- form), QAE Sephadex A25 and A-50 (Na.sup.+ form), SP Sephadex C-25 and C-50 (Na.sup.+ form), DEAE Sephacel (Cl.sup.- form), DEAE Sepharose CL-6B (Cl.sup.- form), CM Sephacel CL-6B (Na.sup.+ form) (commercial products of Pharmacia Fine Chemicals AB., Sweden) and the like. It is also possible to use a suitable anion or cation exchange cellulose for the purification.
The substance thus-obtained may contain impurities to a greater or lesser degree, although its IF inducing activity may be sufficient for practical purpose. If desired, it is also possible to reduce the amount of impurities further by combining these treatments. For example, a more than 1000 fold increase in the specific activity (on the basis of the activity present in the extracted solution) may be obtained by combining the gel filtration and ion exchange treatment without difficultly.
Although the physico-chemical characteristics of the active substance obtained by the process of this invention have not yet completely clarified, they are water-soluble, acidic, high molecular weight substances. Their molecular weight ranges distribute over a very wide range of from about 30,000 to about 3,000,000, although their activities are most prevalent within the ranges shown in Table 1. The molecular weights of the active substances of this invention were determined by column chromatography using various gel filtration agents such as the series of Sepharose and Sephacryl (commercial products of Pharmacia Fine Chemicals AB., Sweden) and Bio-Gel (commercial products of Bio-Rad Laboratories Ltd., U.S.) and the like. The results were compared with the corresponding figures obtained by column chromatography using the following reference materials having identified molecular weights:
blue dextran 2000T (*2.times.10.sup.6), .alpha..sub.2 -macroglobulin from horse serum (*8.2.times.10.sup.5), thyroglobulin from bovine thyroid (*6.69.times.10.sup.5), catalase from bovine lever (*2.1.times.10.sup.5), aldolase from rabbit muscle (*1.58.times.10.sup.5), albumin from bovine serum (*6.7.times.10.sup.4), ovalbumin from hen eggs (*4.3.times.10.sup.4), chymotrypsinogen A from bovine pancreas (*2.5.times.10.sup.4), ribonuclease A from bovine panvreas (*4.3.times.10.sup.4) and the like [* standard molecular weight].